Many methods already exist in the field for achieving the above mentioned determination based on the classical techniques of biochemistry. For instance, chemical reactions can be used to detect a given analyte in a number of different ways. Classical systems include titration or reaction with a specific reagent that gives a colored product or precipitate. The requirement for this detection system is that the reagent is in equivalence or in excess, so that the product can be measured by conventional photometry, turbidimetry, colorimetry, etc. The measuring system is chosen according to the magnitude of the signal to be measured. At very low analyte concentrations detection becomes difficult and greater discrimination can be obtained, for example, by concentrating the reaction product locally e.g. by solvent extraction, centrifugation, etc. which may become tedious and costly. However, the above disadvantage was strongly reduced when a practical system for the measurement of biochemical analytes in extremely low concentrations was made available in 1960. This microanalytical system (radioimmunoassay) took advantage of the characteristics of biological systems for molecular recognition (antigen-antibody reactions) and the extreme sensitivity of radioactive measurements (radioactive isotope labelling). An essential feature of this breakthrough was the concept of limited reagent assay with the tracer label used to measure the distribution of the analyte to be measured between the reagent-bound and the free moieties (see for example: Review Paper "The theoretical aspects of saturation analysis" R. P. Ekins in "In vitro procedures with radioisotopes in medicine", International Atomic Energy Agency, June 1970). Although immunoassays were first described as limited reagent assays, equally practical systems were later described for reagent excess methods (see MILES et al. Biochem. J. 108, 611 (1968).
In addition to volumetric and gravimetric analysis, the present methods thus involve highly sensitive methods such as colorimetry, spectroscopy and radio active measurements. However, many of such techniques are now becoming obsolete as they are tedious, require a relatively large quantity of analyte to be accurate, are based on hard to prepare and difficult to store reagents or require expensive and cumbersome equipment and highly skilled operators. Thus, there is a trend now to develop more subtle methods, which require lesser quantities of reagents and which can be performed safely, quickly and accurately by moderately skilled personnel. Among such methods which have been disclosed lately, some involve the use of optical waveguides including the reactant. For analysis, the waveguide is contacted with the analyte in solution whereby a reaction with the reactant on the wave guide occurs with the consequence that the optical properties of the latter are modified. The measurement of such modification then provides the required data for the analyte determination. According to the teaching of some recent references (for instance, U.S. Pat. No. 4,050,895 (HARDY) AND WO No. 81 100 912 (BUCKLES), the guides can consist (BUCKLES) of a porous light transmitting core impregnated with the reactant into which the analyte will diffuse during the reaction. Or, (BUCKLES or HARDY) the waveguide can consist of a non porous light transmitting core (e.g. glass) coated with a porous or permeable sheath impregnated with the reactant and into which the analyte will diffuse. Furthermore, in one specific case applied to immunoassay (HARDY, Example 3), a rod-shaped waveguide is coated with an antibody layer bonded by diphenyldimethoxysilane and reacted with polystyrene latex spheres treated with an antigen. The antigen treated beads will then attach to the guide and modify the light signal output of the latter, which variation is used for the analytical determination.
The above techniques have merit but they can not be readily applied to some typical analyses involving reaction kinetics. Indeed, it is well known that rates may provide essential analytical data, particularly in the case of automated test systems and, since reactions occurring within permeable or porous bodies always involve a preliminary diffusion of the analyte into said body, and since diffusion processes are generally much slower than chemical reactions, the rate of the latter cannot be measured directly; in such a case, only equilibrium data can be obtained. Also in the known prior-art, embodiments are avoided involving the use of a transparent core coated with a reactant sheath with a refraction index smaller than that of the core for the reason that, admittedly, low sensitivity would be expected to result. Indeed in the latter case most of the light signal injected at the input of the guide will travel within the core by a total reflection process and as a result, as is commonly accepted, the interaction of that signal with the reaction products located in the sheath, i.e. outside the core should only be minor. Consequently, care was taken in the prior art that the refractive index of the sheath n.sub.2 (where the reaction takes place) be always larger than that (n.sub.1) of the core for allowing the light injected in the core at the input to be refracted into the sheath and, from that point on, to continue to travel in the sheath right to the output of the guide. However, contrary to some of the prior disclosures, the output signal (the result of the light having been modified by passing through the products of reaction: reactant+analyte within the sheath) will not readily reenter the core and reach the back end thereof (this behavior results from elementary optical principles to be discussed later), and, for the measurements, the output light detector must be located in the very near vicinity of the testing probe (i.e. the back end of the sheath). Such arrangement is not always practical constructionwise, namely when the guide (plus sheath) is dipped in a liquid for measuring an analyte in solution.